Examining the components of your peptide sample with AccuPep QC

Lauren Lu, Ph.D.

October 29, 2015, 9:00-10:00 AM EST

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Custom peptides play an important role in many research applications

When do I need custom peptides?

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Tissue Engineering Hydrogels • stem cells

• wound healing

Vaccine Development HIV • cancer • influenza

• HPV

Drug Discovery Antimicrobials• cancer • diabetes

• neurodegenerative diseases •

immunotherapy

Antibody Generation Phospho-specific antibodies •

non-commercially available

antibodies

Structural biology NMR • protein-protein interactions •

enzyme assays

Drug Discovery siRNA

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Common issues that you might face

• Unable to dissolve peptides with poor solubility

• Improper dissolving way ruining peptide sample

• Unknown contamination ruining your assay

• Low experiment reproducibility from batch to batch

• Non-reproducible results for quantitative experiments

Common challenges with custom

peptides

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For certain types of assays, additional testing is required to

learn more about the contents of your peptide sample:

• Appropriate solvents

• Removal of TFA

• The precise amount of net peptide

• The presence of endotoxin

• The water %

• The pH value

• The residual solvents

• The identification of peptide impurities

• …

How can these issues be addressed?

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Reduces experimental troubleshooting

Increases experiment reliability

Ensure reproducible results

Benefits of analyzing your peptide

content

5

GenScript offers a comprehensive QC service, AccuPep+, to help

you get the most out of your custom peptides

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• Amino Acid Analysis

• Peptide Content Analysis

• Counter-ion Quantification Analysis

• Moisture Content Analysis

Quantification tests

• TFA Removal and Analysis

• Endotoxin Analysis

Toxicity Tests

• Solubility Tests

• pH Test

Other tests

Features of the AccuPep+ Service

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w w w.genscr ipt .com /accupep_qualit y.ht m l

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Quantification Tests:

• Do I really know all the possible components in my peptide sample?

Toxicity Tests:

• What could make my experiment fail?

Other Tests:

• What else can I do to accelerate my experiments?

AccuPep+ service test options

7

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Quantification Tests:

• Do I really know all the possible components in my peptide sample?

Toxicity Tests:

• What could make my experiment fail?

Other Tests:

• What else can I do to accelerate my experiments?

AccuPep+ service test options

8

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Quantification Tests: Methods

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Components Quantification Methods

Target Peptide Amino Acid Analysis;

Peptide Content Analysis (Nitrogen

Element Analysis);

HPLC Analysis Peptide Impurities

Counter-ion (Trifluoroacetate, Acetate

& hydrochloride)

Counter Ion Quantification Analysis

(Ion chromatography)

Water Moisture Content Analysis (Karl

Fischer coulometric titration)

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How much peptide do I have?

10

Target Peptide

Peptidic Impurities

Counter-ion (Trifluoroacetate, Acetate & hydrochloride)

Water

ca.100%

The net peptide content

TFA-

OAc-

Cl-

(Purity)

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How is peptide purity measured?

11

High performance liquid chromatography (HPLC) Purity

The ratio of peak area of

target peptide in relation

to all detected peak area

Peptide Purity: 97%

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Amino Acid Analysis (AAA) is ideal for:

• Determining the amino acid composition

• Determining the precise amount of net peptide in your sample

Protocol:

What is the amino acid composition

of my peptide?

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Hydrolysis Pre-column

derivatization

HPLC UV

Time

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Example AAA report

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Amino acid

(a.a.) residues Theoretical

a.a. number

The measured a.a.

residues concentration

(μmol/ml) Measured a.a. number

Asp/Asn 1 0.1323 1.00

Glu/Gln

Ser

Gly

His

Arg 1 0.1308 1.00

Thr 1 0.1142 0.90

Ala 1 0.1245 1.00

Pro

Tyr

Val 1 0.1431 1.10

Met

Cys

lle

Leu

Phe 1 0.1269 1.00

Trp

Lys

Peptide Sequence: VFNTRA

Tips: 1. Asn and Gln are de-

aminated during hydrolysis

to Asp and Glu.

2. Only highlighted a.a.

residue is stable enough

during hydrolysis to be used

as for peptide content

calculation.

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Alternative way to determine the

net peptide content

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Peptide Content Analysis: determines the precise amount of net peptide

in the gross peptide sample

Method

• Nitrogen Element Analysis

Nitrogen % (Cx Hy NZ On Sm) %

Note: Given that the counter-ions (acetate, trifluoroacetate or hydrochloride) and

the adsorbed water do not contain nitrogen, Nitrogen element analysis can be

use for the net peptide content measurement.

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How to calculate the net peptide

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Delivered (Gross) Weight:10 mg

Purity: 97%

Peptide content: 40% (AAA)

10 mg * 40% = 4.0 mg

Delivered peptide

Net peptide weight:

Exact amount of target peptide:

With the purity and peptide content as determined by AAA, you can calculate the net peptide weight and exact amount of your peptide

4.0 * 97% = 3.88 mg

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Other constituents in your sample

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Target Peptide

Peptidic Impurities

Counter-ion (Trifluoroacetate, Acetate & hydrochloride)

Water

ca.100%

The net peptide content

TFA-

OAc-

Cl-

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Common counter-ion types

• Trifluoroacetate (main type for most delivered peptides)

• Acetate/Formate/Hydrochloride

Counter-ion sources

• Peptide cleavage and purification

• Counter-ion exchange

Counter-Ion Types and Sources

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TFA

TFA-

Free acid

Salt

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Counter Ion Quantification Analysis

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Counter Ion Quantification Analysis is useful for:

• Cellular assays

• Active pharmaceutical ingredients (APIs)

• Manufactured products

Method

• Ion chromatography

TFA-

OAc-

Cl-

Time

Conductivity

R K

H

NH3+

TFA-

TFA-

TFA-

TFA-

TFA-

OAc-

Cl-

Trifluoroacetate

Acetate

Hydrochloride

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How can I estimate the theoretical TFA

amount?

19

NH2

TFA

Peptide molecular weight: 1500 Da

TFA molecular weight: 114 Da

Peptide (adducted with counter-ion) molecular weight:

1500+114*4 = 1956 Da

The estimated TFA % = 114*4/1956 = 23.3 %

R K H

NH3+

TFA-

TFA-

TFA-

TFA-

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Moisture Content Analysis

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Moisture Content Analysis is useful for hydrophilic peptides

that will retain the most water.

Method

• Karl Fischer Coulometric Titration

• The basis:

I2 reacts quantitatively with H2O while the iodine is

generated directly in the electrolyte by electrochemical means.

The rigorously quantitative relationship between

the electric charge and the amount of iodine generated is used for

high-precision dispensing of the iodine.

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Summary

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Components Quantification Methods

Target Peptide

Usually, 50-80%

Peptide Impurities

Counter-ion (Trifluoroacetate, Acetate

& hydrochloride)

Usually, 30-10%

Dependent on basic a.a. residue

number

Water Usually < 10%,

but could be particularly high for

hydrophilic peptide

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Quantification Tests:

• Do I really know all the possible components in my peptide sample?

Toxicity Tests:

• What could make my experiment fail?

Other Tests:

• What else can I do to accelerate my experiments?

AccuPep+ service test options

22

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TFA removal and analysis

• TFA is a counter anion for normal peptides

• Trace amount of TFA can cause cytotoxicity in cell culture assays

Endotoxin Analysis

• Endotoxins are easily introduced into peptides during any process of

peptide production.

• Small concentrations of endotoxin can decrease cell viability or cause

immune response in cellular assays.

Toxicity test options

23

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TFA Removal and Analysis

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TFA removal method

How to choose counter-ion form (in the point view of production)

R K

H

NH3+

OAc-

OAc- OAc-

OAc- HOAc

R K

H

NH3+

Cl-

Cl- Cl-

Cl- HCl Trifluoroacetate form

Acetate/formate form

Hydrochloride form

HPLC

or Anion exchange resin

OAc-

Cl-

Suitable for unstable amino acids, such as Cys, Met, Gln at N-terminus

Suitable for peptides with low solubility

R K H

NH3+

TFA-

TFA-

TFA-

TFA-

TFA-

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Standard TFA Removal Service

Guaranteed TFA Removal Service

Two Types of TFA Removal Services

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R K

H

NH3+

OAc-

OAc- OAc-

OAc- HOAc

Trifluoroacetate form TFA Content: < 1%

( or HPLC )

R K

H

NH3+

OAc-

OAc- OAc-

OAc- HOAc

Trifluoroacetate form TFA Content: Undetermined,

usually quater of the estimated - 1%

HPLC

Anion exchange resin

( or Anion exchange resin)

R K H

NH3+

TFA-

TFA-

TFA-

TFA-

TFA-

R K H

NH3+

TFA-

TFA-

TFA-

TFA-

TFA-

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Endotoxin Analysis

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Endotoxins (lipopolysaccharides)

• Major components of the cell walls of gram-negative bacteria

• Introduced into custom peptides during peptide production

Method

• Chromogenic Tachypleusamebocyte lysate or Limulus amebocyte

lysate test

• Guaranteed high-sensitivity: 0.005 EU/ml

Peptide solution

ToxinSensorTM

Endotoxin Assay Kits UV

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A variety of applications benefit from TFA removal or

endotoxin analysis:

Which service is best for me?

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Application Method

Analytical analyses that are influenced by TFA

ions:

- Infrared (IR) spectroscopy

- Circular dichroism (CD) spectroscopy

TFA removal and analysis

Cell culture assays TFA removal and analysis

Cosmetics and pharmaceutical applications TFA removal and analysis

Cell culture assays sensitive to endotoxin or

prone to immune responses

Endotoxin analysis

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Even trace amounts of TFA can cause cytotoxicity in cell

culture assays

Case study: effect of TFA on cell

culture

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Peptides containing TFA at

concentrations ranging from 10-9 to 10-7M

were supplemented to osteocyte and

bone cultures.

Viability was assessed by [3H]thymidine

incorporation after 24 hours

[TFA]

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Quantification Tests:

• Do I really know all the possible components in my peptide sample?

Toxicity Tests:

• What could make my experiment fail?

Other Tests:

• What else can I do to accelerate my experiments?

AccuPep+ service test options

29

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Solubility Test

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Hydrophobic peptides

• Containing > 50% hydrophobic amino acid

• Length > 5 a.a.

Hydrophobic :

Ala (A),Trp (W), Leu (L), Ile (I), Phe (F), Met (M), Val (V), Pro (P)

Basic:

Arg (R), His (H), Lys (K)

Polar uncharged:

Asn (N), Cys (C), Gly (G), Gln (Q), Ser (S), Thr (T), Tyr (Y)

Acidic:

Asp (D), Glu (E)

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What is included in your solubility report?

Components of the solubility report

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Solvent Results

(Dissolved or Undissolved) Gross Peptide Concentration

Ultrapure water Undissolved N/A

PBS(pH 7.1±0.1) Dissolved ≤ 1mg/ml

DMSO Dissolved ≤ 10mg/ml

Others* N/A N/A

Acidic a.a.

Polar uncharged a.a.

Hydrophobic a.a.

Benefits:

Reduces troubleshooting

Saves time and peptide products

Particularly useful for peptide libraries

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Why different pH value

• Free TFA acid may be present

• Different counter ions may result in various pH values of

peptide solutions

pH Testing

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High pH

R K

H

NH3+

OAc-

OAc- OAc-

OAc- HOAc

Low pH

R K H

NH3+

TFA-

TFA-

TFA-

TFA-

TFA-

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Conclusions

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Amino Acid Analysis

Peptide Content Analysis

Counter Ion Quantification Analysis

Moisture Content Analysis

Solubility Test

pH Test

TFA Removal and Analysis

Endotoxin Analysis

Quantifying each component Removing toxins

Making sample prep easier

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Click on the quote button

Input your desired peptide

sequence, purity, quantity

and modification

Check the box to receive a

free solubility test

Select a TFA Removal option (guaranteed or

standard)

In the comments

section, request any of

the optional QC services

How can I request these services?

34

To learn more, visit

www.genscript.com/accuprep_quality.html

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Peptide services at GenScript

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Standard peptide synthesis • Starting at $3.2/AA • Up to 200 AA • Mg to kg quantity

Peptide Library • Standard and micro-scale

quantity • Flexible purity options • Peptide pooling available

Peptoid Synthesis • Proteolytic resistant

peptidomimetics • Cost-effective, fast

turnaround

Click Peptide Synthesis • O-acyl bond incorporation

technology

• Increased peptide stability

Cosmetic Peptide Synthesis • High batch-to-batch

reproducibility • High capacity

www.genscript.com/peptide-services.html

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Thank you for your participation

We wish you success with your research

Large scale genome editing for metabolic engineering of E. coli– Yifan Li, Ph.D.

November 5, 2015, 9:00 AM or 2:00 PM EST

Optimizing soluble protein expression: codon optimization, RBS design and expression vector design– Rachel Speer, Ph.D.

November 11, 2015, 9:00 AM or 2:00 PM EST

Antibody Drug Development: challenges & solutions – Liusong Yin, Ph.D.

November 18, 2015, 9:00 AM EST

Register for other webinars in the GenScript Webinar Series or download past webinars at

http://www.genscript.com/webinars.html

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Q&A

37

If you have any other questions, visit www.genscript.com/faq_for_peptide Or email: Lauren.Lu@genscript.com or laura.geuss@genscript.com